Design, synthesis and biological evaluation of 2-Phenyl-4H-chromen-4-one derivatives as polyfunctional compounds against Alzheimer’s disease

Article abstract of DOI:10.1007/s00044-017-2078-4

Polyfunctional compounds comprise a novel class of therapeutic agents for the treatment of multi-factorial diseases. A series of 2-Phenyl-4H-chromen-4-one and its derivatives (5a–n) were designed, synthesized, and evaluated for their poly-functionality against acetylcholinestrase (AChE) and advanced glycation end products (AGEs) formation inhibitors against Alzheimer’s disease (AD). The screening results showed that most of them exhibited a significant ability to inhibit AChE AGEs formation with additional radical scavenging activity. Especially, 5m, 5b, and 5j displayed the greatest ability to inhibit AChE (IC₅₀ = 8.0, 8.2, and 11.8 nM, respectively) and AGEs formation (IC₅₀ = 55, 79, and 54 μM, respectively) with good antioxidant activity. Molecular docking studies explored the detailed interaction pattern with active, peripheral, and mid-gorge sites of AChE. These compounds, exhibiting such multiple pharmacological activities, can be further taken a lead for the development of potent drugs for the treatment of Alzheimer’s disease.

Full text of DOI:10.1007/s00044-017-2078-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2078-4
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of 2-Phenyl-4H-
chromen-4-one derivatives as polyfunctional compounds against
Alzheimer’s disease
Manjinder Singh1 Maninder Kaur1 Bhawna Vyas2 Om Silakari1
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Received: 1 June 2015 / Accepted: 15 September 2017
© Springer Science+Business Media, LLC 2017
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Abstract Polyfunctional compounds comprise a novel
class of therapeutic agents for the treatment of multi-
factorial diseases. A series of 2-Phenyl-4H-chromen-4-
one and its derivatives (5a–n) were designed, synthesized,
and evaluated for their poly-functionality against acet-
ylcholinestrase (AChE) and advanced glycation end pro-
ducts (AGEs) formation inhibitors against Alzheimer’s
disease (AD). The screening results showed that most of
them exhibited a significant ability to inhibit AChE AGEs
formation with additional radical scavenging activity.
Especially, 5m, 5b, and 5j displayed the greatest ability to
inhibit AChE (IC50 = 8.0, 8.2, and 11.8 nM, respectively)
and AGEs formation (IC50 = 55, 79, and 54 µM, respec-
tively) with good antioxidant activity. Molecular docking
studies explored the detailed interaction pattern
with active, peripheral, and mid-gorge sites of AChE.
These compounds, exhibiting such multiple pharmacolo-
gical activities, can be further taken a lead for the devel-
opment of potent drugs for the treatment of Alzheimer’s
disease.
Keywords AChE inhibitor Alzheimer’s disease
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●
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Antioxidants Flavonoids AGEs Flavone
Abbreviations
ChE
Cholinesterase
AD
Alzheimer’s disease
AChE
AGEs
CAS
PAS
ACh
FDA
OS
Acetylcholinesterase
Advanced glycation end products
Catalytic active site
Peripheral anionic site
Acetylcholine
Food and drug administration
Oxidative stress
Reactive oxygen species
ROS
Aβ
β-amyloid
RAGE
DPPH
EWG
Receptor for AGEs
1,1-diphenyl-2-picryl-hydrazyl
Electron withdrawing groups
Introduction
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2078-4) contains supplementary
material, which is available to authorized users.
Alzheimer’s disease (AD), a neurodegenerative disorder
associated with a progressive loss of cognitive functions is
emerging as one of the greatest health threat of the 21st
century affecting almost 50% of adults over the age of 85
(Bishop et al. 2010). Several hypotheses that aim to explain
the initiation and progression of the AD, including choli-
nergic hypothesis, amyloid hypothesis, tau hypothesis, cal-
cium hypothesis, oxidative stress (OS) induction, advanced
glycation end products (AGEs) induction, iron deregulation,
mitochondrial dysfunction, etc., have been proposed. In the
* Om Silakari
omsilakari@gmail.com
1
Molecular Modeling Lab, Department of Pharmaceutical Sciences
and Drug Research, Punjabi University, Patiala, Punjab 147002,
India
2
Department of Chemistry, Punjabi University, Patiala, Punjab
147002, India

Med Chem Res
light of various complex mechanisms and hallmarks
involved in the pathogenesis of AD, it has been recognized
as a complex neurodegenerative disorder with a multifaceted
pathogenesis (Singh et al. 2016) (Fig. 1). Due to the
pathological complexity, to date, no agent has proved to be
considerably effective and the treatment options are limited
for the clinicians. Thus, novel treatment strategy for AD is
the biggest medical need for medical science and research.
The currently available anti-AD developed according to
the reductionist paradigm of “one-molecule-one-target,” has
turned out to be palliative rather than curative. Thus, drug
molecule that can act as multiple targets in neurotoxin
cascades offers new hopes toward curing AD (Schmitt et al.
2004). Such multi-targeted agents can be developed with
superior efficacy and safety profiles (Morphy and Rankovic
2005; Youdim and Buccafusco 2005).
hydrolysis at the cholinergic synapses, and therefore AChE
inhibitors could increase the levels of ACh in AD patients
through the inhibition AChE and thus relieve some symp-
toms experienced by AD patients (Singh et al. 2013). Till
date, acetylcholinesterase inhibitors (AChEIs) are the only
clinical used drugs for the treatment of AD (Fig. 2). Besides
several diverse hallmarks AD brains display constant evi-
dence of oxidative damage, which induces injury in the most
cellular macromolecules of AD brain including nucleic acids,
proteins, and lipids. These findings support the “OS
hypotheses of AD,” in which reactive oxygen species (ROS)
play a key role in the onset and progression of AD (Mar-
kesbery 1997; Melo et al. 2003). Thus, drugs aimed at
clearing or preventing the formation of the free radicals may
be useful for the management of AD.
Moreover, the AGEs’ (senescent macroprotein deriva-
tives, formed through the non-enzymatic glycation called
the “Maillard reaction”) interact with a receptor for AGEs
and provoke the generation of ROS and vascular inflam-
mation, and consequently alters the various gene expres-
sions in several types of cells, which could contribute to the
Cholinergic hypothesis, which proposed that the extensive
decrease of acetylcholine (ACh) leads to cognitive
and memory deficits associated in AD patients represents one
of the conventional hypothesis related to AD. Acet-
ylcholinesterase (AChE) is the key enzyme for ACh
Fig. 1 Role of acetylcholinesterase, AGEs, and ROS in the patho-
physiology of AD: AChE—acetylcholinesterase; ACh—acetylcholine;
Aβ—β-amyloids; AGEs—advanced glycation end products; ApoE4—
apolipoprotein E; RAGE—receptor for AGEs; ROS—reactive oxygen
species;.OH—hydroxyl radicals; H2O2—hydrogen peroxide; O2.-—
superoxide anions; Ca2+—calcium; NFκB—nuclear factor kappa-
light-chain-enhancer of activated B cells

Med Chem Res
Result and discussion
To develop a convincing drug candidate for multi-faceted
AD, we commenced structure-based drug design approach
considering 2-Phenyl-4H-chromen-4-one as a privileged
scaffold, which has already been explored for its broad
range of pharmacological properties for the management of
AD. Although most of the synthesized derivatives have
already been reported for various targets of other different
disease conditions as anticancer, Tankyrases inhibitory,
HSP90 receptor inhibitory, anti-mycobacterium, anti-anxi-
ety, PGE2 and COX-2 inhibtory, HIV-l integrase inhibitory
activities, etc. (Lee et al. 2016; Cabrera et al. 2007; Cár-
denas et al. 2006; Narwal et al. 2013; Lin et al. 2002; Dao
et al. 2004; Fesen et al. 1994) none of these derivatives has
been evaluated against targets of AD like AChE and AGEs
formation. Moreover, till date, no 2-phenyl-4H-chrome-4-
one scaffold-based poly-functional molecule for the man-
agement of AD has been reported to be used in clinical
practice. Thus, it was thought worthwhile to consider this
distinctive scaffold in poly-functional drug design program
for AD.
Fig. 2 FDA approved drugs for the treatment of Alzheimer’s disease
pathological changes of AD as well (Munch et al. 1997;
Yamagishi et al. 2003; Yan et al. 1994) (Fig. 1).
These interrelated hypotheses contribute to the complex
pathogenesis of AD and the compounds that can act at
different levels of the neurotoxic cascade or can modulate
multiple targets simultaneously, offer new hopes toward
curing AD. In the present study, flavone-based polyfunc-
tional agents modulating ACh levels, having antioxidant
potential along with AGEs formation inhibitory activity has
been developed as novel therapeutics for the treatment of
AD (Bolognesi et al. 2009).
Chemistry
The synthesis of the 2-Phenyl-4H-chromen-4-one deriva-
tives 5(a–n) was performed according to the
Baker–Venkataraman rearrangement reaction with slight
modifications depending on the stability and reactivity of
the reagents used. The synthetic methodologies employed to
develop intermediates (3(a–n)) and target compounds (5
(a–n)) are outlined in Scheme 1. The Baker–Venkataraman
rearrangement is one of the fundamental reactions that
involves conversion of o-hydroxyacetophenone into phe-
nolic ester, which undergoes an intramolecular Claisen
condensation in the presence of a base to form β-diketone
(Baker 1933; Mahal and Venkataraman 1934). The com-
pounds were dried, and then purified on silica columns
using hexane:ethyl acetate (6:4) as solvent. The structures
and purities of the target compounds were confirmed by IR,
1H NMR, and mass analysis.
Selection of appropriate privileged scaffold for designing
of polyfunctional drug is a crucial step in the search of
clinical candidates for the treatment of AD. Flavonoids
having 2-Phenyl-4H-chrome-4-one scaffold are well-known
natural compounds possessing a broad range of biological
activities related to AD, such as neuroprotective effect
(Lim et al. 2007), AChE inhibitory activity (Jung and
Park 2007), Aβ fibril formation inhibitory activity (Kim
et al. 2005), antioxidant properties (Zhu et al. 2007),
AGEs formation inhibitory activity, etc. (Singh et al.
2014). Above-mentioned reports and natural antioxidant
potential of flavonoids make them suitable scaffold for
designing of polyfunctional drugs for the management of
AD. In the present study, various derivatives were
designed by exploring the different positions of ring-A and
ring-B of 2-phenyl-4H-chromen-4-one scaffold to improve
AChE and AGEs formation inhibitory activities with
retention of radical scavenging effects. Thus, a series of
polyfunctional flavone derivatives was synthesized in short
steps and were tested for different biological activities.
Moreover, the docking analysis was also employed to
understand interactions of these derivatives with the active
site of AChE.
Evaluation of biological activity
1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging
activity studies
All the compounds (5a–n) were tested for free radical
scavenging activity by using DPPH assay at 517 nm. The
DPPH scavenging activities of test compounds are sum-
marized in Table 1. Most of the compounds (5n, 5l, 5b, 5k,
5m, 5h, 5c, 5i, and 5j) exhibited radical scavenging activity
compared to ascorbic acid (Pi et al. 2008; Fang et al. 2008).

Med Chem Res
Scheme 1 Synthesis of 2-phenyl-4H-chromen-4-one derivatives (5a–n) with reagents and reaction conditions
Advanced glycation end-products formation inhibitory
activity
exhibited significant inhibitory activity. However, the com-
pounds 5h, 5f, 5b, 5d, 5a, 5c, 5g, and 5e (IC50 value ranging
from 65.5 0.52 to 148.5 3.41 μM) exhibited lower anti-
glycation activity as compared with the standard drug.
As evident from the results, among all the synthesized
compounds, the compounds with hydroxyl substitution
(5k–5n) were more active than the derivatives that lack
hydroxyl group (5a–5j). Interestingly, di-hydroxyl-
substituted derivative (5l) was found to be more active
than the mono hydroxyl-substituted derivatives (5n, 5k, and
5m). Additionally, the derivatives with the electron releas-
ing group at 7th position of ring-A showed higher activity
(5n) than the derivatives with the electron withdrawing
group at 7th position of ring-A (5m).
All the synthesized compounds 5a–n were subjected to
in vitro AGEs formation inhibitory activity using the method
reported by Matsuura et al. with slight modification (Mat-
suura et al. 2002). The potential of the compounds 5a–n to
inhibit AGEs formation is summarized in Table 1. All the
synthesized compounds showed considerable AGEs forma-
tion inhibitory activity as the fluorescence of AGEs was
shown to be remarkably reduced by all the synthesized
compounds. Among the various synthesized compounds, 5l
(IC50 = 33.0 1.91 μM) was found to be 1.2 folds more
potent than the standard drug aminoguanidine (IC50 = 40.54
2.01 μM). The compounds 5n, 5k, 5j, 5m, and 5i (IC50
value ranging from 42.0 1.82 to 58.0 1.71 μM) also
Moreover, the compounds having electron withdrawing
groups (EWG) at the meta position of ring-B (5f, 5b, and

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Med Chem Res
5d) showed high AGEs formation inhibitory activity as
compared to compounds with EWG at the para position
(5c, 5g, and 5e), as well as a compound with no substitution
(5a). Besides, the electron releasing groups of ring-B (5i
and 5h) showed significant inhibitory potential than the
compounds having electron withdrawing group (5b–g).
Also, the di-nitro-substituted derivative (5j) showed better
activity than mono nitro-substituted derivative (5b and 5c).
Thus, it can be concluded that flavone derivatives exhibited
significant AGEs formation inhibitory activity that varies by
different substitution patterns around the core structure.
derivative (5c). The flavone derivatives with hydroxyl groups
substituted at ring-A (5k–5n) showed good AChE activity
along with strong radical scavenging properties.
Molecular docking studies
To investigate the binding patterns of synthesized 2-phenyl-
4H-chromen-4-one derivatives in the active site of AChE
enzyme, molecular docking studies were carried out for 5m,
5b, and 5j that showed good anti-cholinesterase activity.
These compounds showed good fit in the active site by
interacting with both catalytic site and PAS simultaneously.
In compound 5m, the hydroxyl group substituted at seventh
position of the ring A showed hydrogen bonding interaction
with Ser200 amino acid residue and His440 amino acid
residue of the catalytic triad. The His440 amino acid residue
also showed π–π stacking with the ring A. Additionally, the
“O“ of the ring C showed hydrogen bonding interaction with
the Tyr121 amino acid residue of the PAS (Fig. 3). In
compound 5b, ring C showed π–π stacking with Trp279
amino acid residue of PAS; ring A showed π–π stacking
with Tyr334 amino acid residue of PAS and with the
Phe330 amino acid residue of catalytic triad. In addition to
π–π stacking, hydrogen bonding interaction was observed
between the carbonyl “O” of ring C and Phe288 and Arg289
amino acid residues of catalytic triad. Also, the nitro group
substituted at third position of ring B showed two hydrogen
bonding interactions with the Tyr121 amino acid residue of
PAS (Fig. 4). Similarly, in compound 5j, ring A showed π–
π stacking with His440 amino acid residue of catalytic triad.
Additionally, it showed two hydrogen bonding interactions,
one between the “O“ of ring-C and Tyr121 amino acid
residue of PAS, and another between “O” of nitro group and
Phe288 amino acid residue of catalytic triad (Fig. 5).
In vitro AChE inhibition studies
The AChE inhibitory activity of all the synthesized com-
pounds was tested in vitro, according to the modified Ell-
man’s method (Ellman et al. 1961), using the rat brain
homogenate, whereas donepezil was taken as a reference
drug. The results are summarized in Table 1. Three inde-
pendent experiments were performed to evaluate the AChE
inhibitory effect of the synthesized compounds.
As shown in Table 1, among all, three compounds showed
higher (5m, 5b, and 5j, IC50 = 8.0, 8.2 and 11.2 nM,
respectively) AChE inhibitory activity than donepezil (IC50
= 12.7 nM). The compounds 5n, 5g, 5h, and 5i also showed
significant AChE inhibitory activity. The compound 5m with
hydroxyl substituent at seventh position of ring-A and fluoro
at the fourth position of ring-B showed highest AChE
activity, i.e., IC50 8.0 nM. Removal of the hydroxyl group
resulted in 2–3 fold decrease in activity (5h, 5g, and 5e, IC50
= 16.0, 24.0, and 25.0 nM, respectively). Replacing of the
electron withdrawing group of compound 5m with an elec-
tron releasing group (–OCH3) and introduction of a hydroxyl
group at position seventh of ring A result in decrease activity
(5n, IC50 = 35.0 nM). Also, compounds 5h and 5i, having a
methoxy group at the ring-B showed significant AChE
inhibitory potency (IC50 = 16.0 nM and 24.0 nM, respec-
tively), indicating the importance of methoxy group in ring-B
for AChE inhibition effects.
As evident from docking analysis, the compounds
showed hydrogen bond, π–π (aromatic) and hydrophobic
interactions with both CAS and PAS of AChE concurrently.
Additionally, it was observed that meta-substituted com-
pounds (5d and 5f, IC50 = 31.0 and 37.0 nM, respectively)
showed less AChE inhibitory activity as compared to para-
substituted compounds (5g and 5e, IC50 = 24.0 and 25.0 nM,
respectively), except meta-nitro-substituted derivative (5b,
IC50 = 8.2 nM) that showed 4-fold higher activity as com-
pared to para-nitro-substituted derivative 5c (IC50 = 32.0
nM). This exception may be due to the reason that compound
5b showed π–π stacking interaction with the conserved
residue Trp279, major binding component of peripheral
anionic site (PAS) and important interaction with Phe330,
Phe288, and Arg289 of catalytic anionic site (CAS). Also, di-
nitro-substituted derivative (5j) showed good AChE activity
with IC50 11.8 nM, than mono para-nitro-substituted
Conclusion
In the present study, 2-Phenyl-4H-chromen-4-ones deriva-
tives have been developed as potential poly-functional anti-
Alzheimer’s agents. Most of the synthesized compounds
exhibited potent AChE inhibitory activity with good radical
scavenging and AGEs product formation inhibitory activity.
The compounds 5m, 5b, and 5j (IC50 = 8.0, 8.2, and 11.8
nM, respectively) displayed higher potency for AChE
inhibition. Furthermore, molecular modeling study indi-
cated that compounds 5m, 5b, and 5j simultaneously bind
with both CAS and PAS of the active site gorge. Addi-
tionally, these compounds had significant capacity to absorb
free radicals and inhibit the AGEs formation. Thus,

Med Chem Res
Fig. 3 Docked pose of interactions of flavone derivative (5m) with AChE (1EVE)
Fig. 4 Docked pose of interactions of flavone derivative (5b) with AChE (1EVE)
compounds 5m, 5j, 5l, and 5n have been found to be
maximally potent polyfunctional molecules. These poly-
functional attribute of the 2-Phenyl-4H-chromen-4-ones
make them potential candidates for the development of
drugs for AD. However, further detailed investigations of
mechanisms involved in these activities may establish their
specific therapeutic usefulness.
any purification. The completion of each reaction was
monitored by thin layer chromatography (DC-Alufolien
(20 × 20 cm) Kieselgel 60 F254 chromato plates) using
hexane:ethylacetate (6:4) as a TLC development solvent
system. All final compounds were purified on silica col-
umns while all intermediates were purified by recrystalli-
zation from appropriate solvent. The melting point were
recorded on a Labtronics digital automatic melting point
apparatus and uncorrected, IR spectra were recorded on a
Bruker (Alpha E) FT/IR spectrophotometer, 1H-NMR
spectra were recorded on a Bruker Advance II 400 MHz
NMR spectrometer using chloroform (CDCl3) or dime-
thylsulfoxide (DMSO-d6) as solvent and tetramethylsilane
(TMS) as an internal standard. Proton chemical shifts
are expressed in parts per million (ppm). Mass spectra
Experimental section
Chemistry
All chemicals were procured from Sigma Aldrich Co., SD
fine, Loba chemicals and were 99% pure, thus used without

Med Chem Res
Fig. 5 Docked pose of interactions of flavone derivative (5j) with AChE (1EVE)
(ESI-MS, positive) were recorded with a Waters, Q-TOF
micromass (LC-MS).
2-Phenyl-4H-chromen-4-one derivatives (5a–n)
The diketones (4a–n), in the presence of glacial acetic acid
and concentrated sulfuric acid, were refluxed in water bath
for 1 h, to achieve their cyclized products. Then, the reac-
tion mixture was poured onto crushed ice with vigorous
stirring, resulting in the precipitation of the 2-phenyl-4H-
chromen-4-one and its derivatives. The product was filtered,
thoroughly washed with water to make it free from acid and
dried. Then, final compounds (5a–n) were purified on silica
columns using hexane:ethyl acetate (6:4) as solvent.
o-Benzoyloxyacetophenones (3a–n)
At first, the substituted o-benzoyloxyacetophenones (3a–n)
were synthesized by stirring the mixture of substituted o-
hydroxyacetophenone (0.1 mol) and substituted benzoyl
chloride (0.15 mol) in dry pyridine. During the reaction, the
mixture evolved spontaneous heat, and after about 15 min
when the temperature comes down to room temperature, the
mixture was poured with constant stirring into 3% hydro-
chloric acid containing crushed ice resulting in the pre-
cipitation of solid residue. The solid residue was then
filtered and washed with methanol followed by water. The
filtered residue was air dried and re-crystallized from
methanol resulting in the white precipitates of substituted
o-benzoyloxyacetophenone, yield 90%.
2-Phenyl-4H-chromen-4-one (5a) White, crystalline, yield
1
91%, m.p.: 93–96 °C (reported 96–97 °C); H-NMR (400
MHz, DMSO-d6, δ ppm): 6.88 (1H, s), 7.48 (1H, m),
7.55–7.59 (3H, m), 7.68 (1H, d, J = 8.24 Hz), 7.75–7.80
(1H, m), 8.01 (2H, m) 8.13 (1H, dd, J = 1.68, 7.96 Hz). IR:
(C=O) 1645 cm−1 (s), (C–O) 1375–1128 cm−1. MS (ESI)
m/z = 223.07 [M+H]+, Rf value: 0.51 (hexane:ethyl acet-
ate, 6:4).
o-Hydroxydibenzoylmethane (4a–n)
To
a
warm solution of substituted o-benzoylox-
yacetophenone (3a–n) in dry pyridine, hot pulverized 85%
potassium hydroxide was added followed by mechanical
stirring for 15 min resulting in the gradual appearance of
yellow precipitates of potassium salt of substituted o-
hydroxydibenzoylmethane (4a–n). The reaction mixture
was brought down to room temperature and subsequently
acidified with 100 mL of 10% acetic acid to desalt the
compounds. The light-yellow precipitates of diketone
(4a–n) were filtered and dried, yield 85%.
2-(3-Nitrophenyl)-4H-chromen-4-one (5b) Light yellow,
crystalline solid, yield 90%, m.p.: 251–256 °C; H-NMR
1
(400 MHz, CDCl3, δ ppm): 6.92 (1H, s), 7.46–7.50 (1H,
m), 774–7.78 (2H, m), 7.66 (1H, d, J = 7.76 Hz), 8.24–8.27
(2H, dd, J = 1.80, 7.76 Hz), 8.40 (1H, t, J = 2.28 Hz), 8.83
(1H, t, J = 3.68 Hz), IR: (C=O) 1631.49 cm−1 (m), (N=O)
1516, 1372 cm−1 (w), (C–O) 1363–1006 cm−1. MS (ESI)
m/z = 268.07 [M+H]+, Rf value: 0.36 (hexane:ethyl acet-
ate, 6:4).

Med Chem Res
2-(4-Nitrophenyl)-4H-chromen-4-one (5c) Dark gray,
amorphous powder, yield: 86%, m.p.: 294–297 °C. 1H-
NMR (400 MHz, DMSO-d6, δ ppm): 6.85 (1H, s), 7.43
(1H, m), 7.55 (2H, d, J = 8.12 Hz), 7.71 (1H, m), 8.05 (2H,
dt, J = 2.36, 8.92 Hz), 8.18 (2H, dd, J = 1.96, 7.96 Hz),
8.20 (1H, m). IR (KBr pellets): (N=O) 1341, 1518 cm−1
(s), (C=C) 1601 cm−1 (m), (C=O) 1656 cm−1 (s), MS
(ESI) m/z = 268 [M+H]+, Rf value: 0.71 (hexane:ethyl
acetate, 6:4).
ppm): 3.90 (3H, s, –CH3), 6.86 (1H, s), 7.03–7.05 (2H, dd,
J = 2.08, 6.88 Hz), 7.43–7.45 (1H, dd, J = 1.04, 7.16 Hz),
7.58 (1H, dd, J = 0.68, 8.36 Hz), 7.69–7.71 (1H, m),
7.90–7.92 (2H, dd, J = 2.12, 9.0 Hz), 8.23 (1H, dd, J =
1.64, 8.0 Hz), IR: (C=O) 1648 cm−1 (s), MS (ESI) m/z =
253.1 [M+H]+, Rf value: 0.49 (hexane:ethyl acetate, 6:4).
2-(3,4,5-Trimethoxyphenyl)-4H-chromen-4-one (5i) Light
1
gray, solid amorphous, yield 76%, m.p.: 165–168 °C; H-
NMR (400 MHz, CDCl3, δ ppm): 3.91, (3H, s, –CH3), 3.94
(6H, s, –CH3), 6.80 (1H, s), 7.18 (2H, d, J = 12.56 Hz),
7.42–7.46 (1H, m), 7.61 (1H, d, J = 7.84 Hz), 7.70–7.72
(1H, m), 8.81 (1H, dd, J = 1.64, 7.96 Hz), IR: (C=O) 1651
cm−1 (s), MS (ESI) m/z = 313.1 [M+H]+ Rf value: 0.55
(hexane:ethyl acetate, 6:4).
2-(3-Chlorophenyl)-4H-chromen-4-one (5d) Light yel-
lowish brown, amorphous powder, yield 83%, m.p.:
221–227 °C; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.82
(1H, s), 7.42–7.53 (3H, m), 7.60 (1H, d, J = 8.16 Hz), 7.75
(1H, m), 7.80 (1H, d, J = 2.84, 7.64 Hz), 7.93 (1H, t, J =
3.60 Hz), 8.22–8.25 (1H, dd, J = 1.56, 7.96 Hz). IR: (C=O)
1633 cm−1 (s), (C–Cl) 1092 cm−1 (w), (C–O) 1363–1006
cm−1. MS (ESI) m/z = 257 [M+H]+, Rf Value: 0.60
(hexane:ethyl acetate, 6:4).
2-(3,5-Dinitrophenyl)-4H-chromen-4-one (5j) Yellowish
brown, solid amorphous, yield 83%, m.p.: 241–246 °C; 1H-
NMR (400 MHz, DMSO-d6, δ ppm): 7.41 (1H, s),
7.50–7.54 (1H, m), 7.81–7.85 (2H, m), 8.13 (1H, d, J =
7.12 Hz), 9.08 (1H, t, J = 2.0 Hz), 9.24 (2H, d, J = 1.96
Hz), IR: (C=O) 1650 cm−1 (s), MS (ESI) m/z = 312.1 [M
+H]+, Rf Value: 0.60 (hexane:ethyl acetate, 6:4).
2-(4-Chlorophenyl)-4H-chromen-4-one (5e) Off white,
amorphous powder, yield: 70%, m.p.: 191–194 °C (reported
188–190 °C). 1H-NMR (400 MHz, CDCl3, δ ppm): 6.84
(1H, s), 7.44 (1H, m), 7.51 (2H, d, J = 8.64 Hz), 7.58 (1H,
d, J = 7.88 Hz), 7.72 (1H, m), 7.88 (2H, d, J = 8.68 Hz)
8.03 (1H, dd J = 1.96, 6.72 Hz), 8.24 (1H, dd, J = 1.56,
7.92 Hz). IR (KBr pellets): (C–Cl) 752 cm−1(s), (C=C)
1596 cm−1 (m), (C=O) 1655 cm−1 (s). MS (ESI) m/z = 257
[M+H]+, 258 [M+2], Rf value: 0.77 (hexane:ethyl acetate,
6:4).
7-Hydroxy-2-phenyl-4H-chromen-4-one
(5k) White,
crystalline solid, yield: 65%, m.p.: 242–246 °C (reported
245–247 °C). 1H-NMR (400 MHz, DMSO-d6, δ ppm): 6.76
(1H, s), 6.90 (1H, dd, J = 2.20, 8.68 Hz), 6.95 (1H, d, J =
2.16 Hz), 7.53–7.57 (3H, m), 7.91–7.99 (2H, m), 7.92 (1H,
d, J = 8.72 Hz), 10.60 (1H, s). IR (KBr pellets): (C=C)
1604 cm−1 (m), (C=O) 1658 cm−1 (s), (O–H) 3470 cm−1
(w). MS: m/z: 239.10 [M+1], Rf value: 0.24 (hexane:ethyl
acetate, 6:4).
2-(3-Fluorophenyl)-4H-chromen-4-one (5f) Gray, amor-
phous powder, yield 78%, m.p.: 211–215 °C; 1H-NMR
(400 MHz, CDCl3, δ ppm): 6.83 (1H, s), 6.90–6.93 (1H, dd,
J = 2.20, 8.72 Hz), 6.98 (1H, d, J = 2.16 Hz) 7.33 (1H, m),
7.54–7.59 (2H, m), 7.75–7.79 (1H, dt, J = 3.96, 7.92 Hz),
7.83 (1H, d, J = 7.88 Hz), 7.92 (1H, d, J = 8.72 Hz), IR:
(C=O) 1621 cm−1 (s), (C–F) 1250 cm−1 (s), (C–O)
1382–1001 cm−1 MS (ESI) m/z = 241.08 [M+H]+, Rf
value: 0.40 (hexane:ethyl acetate, 6:4).
5,7-Dihydroxy-2-phenyl-4H-chromen-4-one
(5l) Light
yellowish, amorphous powder, yield: 72%, m.p.: 288–290 °C
1
(reported 284–289 °C). H-NMR (400 MHz, DMSO-d6, δ
ppm): 6.21 (1H, s), 6.46 (1H, d, J = 2.04 Hz), 6.79 (1H, s),
7.52–7.59 (3H, m), 7.98 (2H, dd, J = 1.44, 7.48 Hz), 10.63
(1H, s), 12.75 (1H, brs).IR (KBr pellets): (C=C) 1598 cm−1
(m), (C=O) 1652 cm−1 (s), (O–H) 3470 cm−1 (w). MS:
m/z: 255.10 [M+1]+, Rf value: 0.53 (hexane:ethyl acetate,
6:4).
2-(4-Fluorophenyl)-4H-chromen-4-one (5g) White, amor-
phous powder, yield: 82%, m.p.: 143–145 °C (reported
148–150 °C). 1H-NMR (400 MHz, CDCl3, δ ppm):6.78
(1H, s), 7.26 (2H, m), 7.46 (1H, m), 7.58 (1H, dd, J = 0.92,
9.16 Hz), 7.72 (1H, m), 7.93 (2H, m) 8.25 (1H, dd, J =
1.84, 7.80 Hz). IR (KBr pellets): (C–F) 1039 cm−1 (w),
(C=C) 1601 cm−1 (s), (C=O) 1657 cm−1 (s), MS (ESI) m/z
= 241 [M+H]+, Rf value: 0.54 (hexane:ethyl acetate, 6:4).
2-(4-Fluorophenyl)-7-hydroxy-4H-chromen-4-one (5m)
Grayish brown, amorphous powder, yield 90%, m.p.:
253–256 °C; 1H-NMR (400 MHz, CDCl3, δ ppm): 6.81
(1H, s), 6.90 (1H, dd, J = 2.24, 8.68 Hz), 6.96 (1H, d, J =
4.0 Hz), 7.30–7.35 (2H, m), 7.90 (1H, d, J = 8.72 Hz),
8.06–8.10 (2H, m), 10.66 (1H, s, –OH). IR: (C=O) 1647
cm−1 (s), (C–F) 1226 cm−1 (m), (O–H) 3665 cm−1. MS
(ESI) m/z = 257.1 [M+H]+, Rf value: 0.57 (hexane:ethyl
acetate, 6:4).
2-(4-Methoxyphenyl)-4H-chromen-4-one (5h) Light yel-
low, solid amorphous, yield 88%, m.p.: 155–158 °C
(reported 154–156 °C); 1H-NMR (400 MHz, CDCl3, δ

Med Chem Res
7-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one
All experiments were performed in triplicate (Prathapan
et al. 2012).
(5n) Brown black, amorphous solid, yield 91%, m.p.:
1
231–236 °C; H-NMR (400 MHz, DMSO-d6, δ ppm): 3.87
(3H, s, –CH3), 6.69 (1H, s), 6.87–6.90 (1H, dd, J = 2.20,
8.68 Hz), 6.94 (1H, d, J = 2.16 Hz), 7.05–7.08 (2H, d, J =
2.96 Hz), 7.89 (1H, d, J = 8.68 Hz), 7.94–7.96 (2H, dd, J =
1.88, 7.0 Hz), 10.62 (1H, s, –OH). IR: (C=O) 1652 cm−1
(s) (O–H) 3701 cm−1, MS (ESI) m/z = 269.1 [M+H]+, Rf
value: 0.53 (hexane:ethyl acetate, 6:4).
In vitro inhibition studies on AChE
AChE inhibitory activity was measured by the spectro-
photometric method with slight modification; rat cortex
homogenate was used as the source of AChE. For assay of
AChE inhibitory activity, a reaction mixture containing 100
μl acetylthiocholine iodide 0.075 M/l, 100 μl sodium phos-
phate buffer (0.1 M/l, pH 7.4), 20 μl homogenate or serum
and different concentrations of test compounds 20 μl were
incubated at 37 °C for 15 min. The reaction was terminated
by adding 50 μl 3% sodium lauryl sulfate, then, 50 μl of
0.2% of 5,5′–dithio-bis-(2-nitrobenzoic acid) was added to
produce the yellow anion of 5-thio-2-nitro-benzoic acid.
The values of IC50 were calculated by UV spectroscopy
from the absorbance changes at 450 nm. Donepezil was
applied as positive drug. All samples were assayed in
triplicate.
Pharmacology and molecular docking
DPPH radical scavenging activity method
The stable 1, 1-Diphenyl-2-picryl hydrazyl radical (DPPH)
was used for determination of free radical-scavenging
activity of the test compounds. The 0.1 mM solution of
DPPH in methanol (39.4 mg in 1000 ml) was freshly pre-
pared. Different concentrations of test compounds were
added to an equal volume of methanol solution of DPPH.
After 30 min at room temperature, the absorbance was
recorded at 517 nm. IC50 values denote the concentration of
the sample, which is required to scavenge 50% of DPPH
free radicals. IC50 value was determined from the plotted
graph of scavenging activity against the different con-
centrations of test compounds. Ascorbic acid was applied as
positive drug (Blois 1958).
Molecular docking studies
Among all the synthesized derivatives, compounds showing
good anti-cholinesterase activity was sketched and cleaned
in maestro molecular modeling workspace followed by
energy minimization in “ligprep“ program of Schrödinger
software using OPLS_2005 force field at pH of 7.4 (Ligprep
2011). The X-ray crystallographic structure of the AChE
complex with donepezil (PDB code 1EVE) was obtained
from the Protein Data Bank and optimized for docking
analysis (Kryger et al. 1999). The optimization protocol
includes the addition of hydrogen atoms, deletion of water
molecules, completion of bond orders, assignment of
hydrogen bonds, and complex minimization to RMSD of
0.20 Å using OPLS_2005 force field. The studied molecules
were docked into the active site of the protein using extra
precision (XP) docking mode of “glide“ program (Glide
2010; Friesner et al. 2004).
In vitro advanced glycation end-product (AGEs) formation
inhibitory activity
The assay for the ability of the flavone to inhibit the
glucose-mediated protein glycation and the development of
fluorescent AGEs was performed. Different concentrations
of various compounds were prepared by dissolving in
DMSO. Antiglycation assay was performed according to
the methods reported by Matsuura and colleagues with
slight modification. In all experiments, about 500 µl of
bovine serum albumin (1 mg/ml final concentration) was
incubated with 400 µl of glucose (500 mM) in the presence
of 100 µl of test compounds, aminoguanidine or phosphate
buffer saline as control buffer at different concentrations.
The reaction was allowed to proceed at 60 °C for 24 h and
thereafter reaction was stopped by adding 10 µl of 100% (w/
v) trichloroacetic acid (TCA). Then the mixture was kept at
4 °C for 10 min before subjecting to centrifugation at
15,000 rpm. The precipitate was redissolved in 1 ml alkaline
PBS (pH 10) and immediately quantified for the relative
amount of glycated BSA based on fluorescence intensity by
spectrofluorometer LS-55 (Perkin Elmer) at 370 nm (exci-
tation) and 440 nm (emission). Aminoguanidine was used
as a positive control. Percentage inhibition was calculated.
Statistical analysis
Data were analyzed by one-way ANOVA followed by
Newman–Keuls multiple comparison test. All statistical
analyses were processed using GraphPad prism software
(version-5.01). Statistical significance was accepted for
P-values of <0.05.
Acknowledgements We acknowledge the financial support from the
“Indian Council of Medical Research (ICMR)”, New Delhi, for pro-
viding us Senior Research Fellowships (ICMR-SRF); Award nos. BIC/
11(11)/2014 and BIC/11(02)/2013.

Med Chem Res
Compliance with ethical standards
Jung M, Park M (2007) Acetylcholinesterase inhibition by flavonoids
from Agrimonia pilosa. Molecules 12:2130–2139
Conflict of interest The authors declare that they have no competing
interests.
Kim H, Park BS, Lee KG, Choi CY, Jang SS, Kim YH, Lee SE (2005)
Effects of naturally occurring compounds on fibril formation and
oxidative stress of beta-amyloid.
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